Cannabis sativa L.) has been used as a health food and folk medicine in China for centuries. In the present study, we sought to define the underlying mechanism by which the extract of Fructus Cannabis (EFC) protects against memory impairment induced by D-galactose in rats. To accelerate aging and induce memory impairment in rats, D-galactose (400 mg/kg) was injected intraperitoneally once daily for 14 weeks. EFC (200 and 400 mg/kg) was simultaneously administered intragastrically once daily in an attempt to slow the aging process. We found that EFC significantly increased the activity of superoxide dismutase, while lowering levels of malondialdehyde in the hippocampus. Moreover, EFC dramatically elevated the organ indices of some organs, including the heart, the liver, the thymus, and the spleen. In addition, EFC improved the behavioral performance of rats treated with D-galactose in the Morris water maze. Furthermore, EFC inhibited the activation of astrocytes and remarkably attenuated phosphorylated tau and suppressed the expression of presenilin 1 in the brain of D-galactose-treated rats. These findings suggested that EFC exhibits beneficial effects on the cognition of aging rats probably by enhancing antioxidant capacity and anti-neuroinflammation, improving immune function, and modulating tau phosphorylation and presenilin expression.Hempseed ( In the past decades, the population birth rates are decreasing, and human lifespan is expanding. As a result, 11% of the world's population is over the age of 60, and this proportion is expected to double by 2050 [Among aging theories, free radical theory is one of the most widely accepted explanations for senescence . This th Cannabis sativa, which has been commonly used as a Chinese traditional medicine and nonstaple food for centuries. It contains a variety of essential fatty acids, such as linoleic acid (ω-6) and alpha-linolenic acid (ω-3) [Hempseed is the fructus ofid (ω-3) . Thus, eid (ω-3) . Hempseeid (ω-3) . In addiid (ω-3) . Hempseeid (ω-3) . HoweverResidents dwelling in Hongshuihe River basin, Guangxi Province, have used hempseed as a popular health food for centuries. In previous studies, we evaluated the cognitive function of longevity population in Hongshuihe River basin by using the psychometric tests of the mini-mental state examination (MMSE) . The resHempseed was purchased in farm product market in Bama County, Guangxi Province, China. D-Galactose was purchased from Sigma-Aldrich . SOD and MDA kits were obtained from Nanjing Jiancheng Bioengineering Institute . Primary antibodies for total tau, phosphorylated tau, and presenilin-1 were purchased from Abcam , and anti-glial fibrillary acidic protein (GFAP) antibody was from Sigma-Aldrich . Alexa Fluor 555 antibody was purchased from Thermo Fisher Scientific . GAPDH was from Good Here Biotechnology . Goat anti-rabbit IgG and goat anti-mouse IgG were purchased from LI-COR Biosciences .Cannabis sativa L.) was identified by Dr. Zhenguo Zhong, a professor of Guangxi University of Traditional Chinese Medicine. EFC was prepared as described previously [Hempseed  with a Lichrospher 100 RP chromatographic column and identified according to mass spectrometry (MS). The mobile phase consisted of acetonitrile and ammonium acetate (12 : 88) at a flow rate of 0.8 ml/min during a run time of 34 minutes and the column temperature was 35°C.Three-month-old Sprague-Dawley (SD) male rats  were obtained from the Experimental Animal Center, Guangxi Medical University (SYXK 2009-0002). The rats were housed in ventilated cages at 24 ± 2°C, with a humidity of 50 ± 10%, under a 12 h light/dark cycle. Standard diet and water were available ad libitum. The experimental protocols were approved by the Institutional Ethical Committee of Guangxi Medical University .After 1 week of acclimatization to vivarium conditions, the animals were screened for the Morris water maze test. Rats that reached the hidden platform within 90 seconds were selected. Forty eligible rats were randomly divided into five groups with 8 rats for each: (1) control group; (2) EFC group (400 mg/kg); (3) D-gal group ; (4) D-gal + EFC (200 mg/kg) group; and (5) D-gal + EFC (400 mg/kg) group. To establish the animal model of aging, rats in the D-gal group were injected intraperitoneally with D-gal at a dose of 400 mg/kg/day for 14 weeks, and the rats in control and EFC (400 mg/kg) groups were injected with an equal volume of normal saline. At the same time, EFC was administered intragastrically once per day for 14 weeks.n = 8 each group) were tested in Morris water maze as described previously [After 14 weeks of drug administration, rats /body weight (g) × 100%.4 rpm for 15 minutes at 4°C, and supernatants were collected for analysis. SOD activities and MDA levels in the hippocampus were analyzed according to the manufacturer's instructions.Hippocampus tissues were prepared in ice-cold physiological saline and centrifuged at 1.2 × 10It is well known that the activation of astrocytes is indicated as increased glial fibrillary acidic protein (GFAP) level. The protocol for immunofluorescent staining has been described previously . SectionThe proteins of hippocampus tissue were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were incubated overnight at 4°C with the primary antibodies appropriately diluted. The secondary antibody was diluted 1 : 3000 in Tris-buffered saline with Tween 20 (TBST). LI-COR Odyssey platform (infrared laser) was used to detect the immune-reactive bands. GAPDH was used as an internal control, and the intensity of the bands was quantified using Odyssey V3.0 software.P < 0.05.Results were analyzed by one-way analysis of variance (ANOVA) with Dunnett's test for multiple post hoc comparisons, and data are expressed as means ± SE (SPSS19.0). Differences were considered statistically significant for ω-6 PUFAs) and omega-3 polysaturated fatty acids (ω-3 PUFAs) in a ratio of 3 : 1. HPLC chromatogram of EFC is shown in It is well known that hempseed is rich in essential fatty acids (EFAs). Therefore, EFC was analyzed by HPLC-MS to identify the components of EFAs. The results show that EFC contains mainly polyunsaturated fatty acids with omega-6 polyunsaturated fatty acids . The organ indices, including heart, liver, thymus, and spleen, in the D-gal model group were significantly lower than those of the control group (P < 0.05)  or EFC treatment (400 mg/kg) improved the general appearance of the rats. The mean body weight in D-gal group notably decreased after 10 weeks, but those of the other groups increased steadily throughout the experiment . Moreove < 0.05) . HoweverP < 0.05, Figures P < 0.05).The Morris water maze test is widely used to evaluate the spatial learning and memory. The navigation training showed a decrease in escape latency and distance to target platform in all groups, which demonstrated that rats in all groups have the ability to learn and to navigate to the goal. There was no difference in escape latencies and distances to hidden platform between groups on Day 1 of training. However, from Day 2 to Day 5, the post hoc comparisons revealed that D-gal model group showed longer escape latencies and distances to find the target platform than the control group . These results indicated that EFC treatment prevented the age-related memory deficits induced by D-gal.At the end of the acquisition training, the hidden platform was removed, and a probe trial was used to assess the reference memory. Rats in D-gal plus EFC (400 mg/kg) and EFC treatment (400 mg/kg) group spent more time in the target quadrant than those in D-gal group . The levels of MDA in the D-gal group in the hippocampus were significantly higher (P < 0.05) than those of the normal control group. Additionally, compared with D-gal group with elevated MDA level, the MDA levels of D-gal plus EFC (400 mg/kg) and EFC treatment (400 mg/kg) group notably reduced (P < 0.05).To assess the effects of EFC on oxidative stress induced by D-gal, we examined SOD activities and MDA levels in the hippocampus. The activities of SOD in the D-gal model group were significantly lower than those of the control group (P < 0.05). However, treatment with EFC significantly reduced the number of GFAP-positive cells in EFC (400 mg/kg) group and D-gal plus EFC (400 mg/kg) group compared with D-gal treated rats , but there was no significant difference in total tau level between groups.To investigate whether EFC has potential neuroprotective effect against brain aging, we examined the levels of presenilin 1 (PS1), total tau, and phosphorylated tau. Western blot analysis indicated that the levels of PS1 and the phosphorylation of tau in the hippocampus of the D-gal administration group were significantly higher than those in the control group  for 8–12 weeks have been widely used to generate a senescence model for oxidative stress and antiaging research , 20. TheWe investigated the learning and memory ability of rats in a Morris water maze. D-gal-treated rats showed significantly longer escape latencies and distances to the hidden platform and less staying time, as well as more times crossing over the location of the previous platform. However, EFC treatment (400 mg/kg) could attenuate these outcomes. These data are in agreement with previous reports which suggested that the improvement of cognition might be the beneficial effect of polyunsaturated fatty acids enriched in the hempseeds , 26.We found that SOD activity was significantly decreased in D-gal treated rats, and MDA content was dramatically increased. However, EFC reversed these changes in the D-gal-treated rats by enhancing the antioxidization capacity. Senescence is the result of oxidative stress, according to the free radical theory of aging . D-gal iAfter 14 weeks of D-gal injection, the heart and liver indices of rats were notably decreased, which implying that aging was accelerated. To the best of our knowledge, the heart and the liver are both important metabolic organs and the function of heart is to provide the pumping pressure that allows blood flow deliver adequate oxygen and nutrients to organs and tissues. The liver is the main part of biological oxidation, participating in glucose, lipid, and protein metabolism as well as immune defense. Meanwhile, the thymus and the spleen indices of rats were significantly decreased, indicating that D-gal can also cause the atrophy of immune organs, as well as the declined immune function, which may be one of the reasons of immune aging. These results support the concept that thymus is the first organ to be sensitive to aging in the body, and thymus atrophy is the most obvious immunologic change in aging . Thus, tAstrocytes are the main components of glial cells in the nervous system, mounting evidence indicated that neuroinflammation is a result of activation of astrocytes which leads to the memory impairment in the D-gal aging mouse model . Our resTo further investigate the possible mechanism of EFC in ameliorating the memory decline in D-gal administration rats, we examined the levels of total tau, phosphorylated tau, and PS1. Tau is a microtubule-associated protein; its main functions are to facilitate the assembly and maintenance of microtubules and to maintain axonal transport in neurons . To the β-peptide (Aβ) production. Alzheimer-associated mutations in PS1 increase production of Aβ42 (the neurotoxic form of Aβ), resulting in deposition of Aβ and senile plaques which is a hallmark of AD [β), PS1 could directly bind tau and GSK-3β to the same region of PS1, which regulates phosphorylation of tau [The present study demonstrated that PS1 expression was markedly higher in the aging model than the control group, and that EFC (400 mg/kg) significantly decreased PS1 expression. The results indicated that treatment with EFC could improve spatial learning and memory in D-gal-treated rats by attenuating tau phosphorylation and by suppressing expression of PS1, which are related to brain aging. Mutations in PS1 are the major cause of the most aggressive form of familial Alzheimer's disease (FAD) which leads to defective learning and impaired memory , 45. In rk of AD . Furtherrk of AD . On the n of tau . Moreoven of tau .In summary, we found that EFC mitigated the features of aging induced by D-gal in rats and improved behavioral performance in Morris water maze. The antiaging effects of EFC include reducing oxidative stress, improving immune function, and inhibiting the activation of astrocytes as well as attenuating tau phosphorylation and suppressing PS1 expression . Therefo


Preoperative identification of patients with high risk of recurrent disease might enable individualized treatment based on patients’ risk profile. The aim of the present work was to investigate the role of metabolic parameters derived from the pre-ablation 18F-FDG PET/CT available. Multivariable cox regression for survival analysis was performed using different models for the metabolic parameters , and total lesion glycolysis (TLG)) corrected for tumour and procedure characteristics. The study endpoints were defined as local tumour progression free survival (LTP-FS), new intrahepatic recurrence free survival (NHR-FS) and extrahepatic recurrence free survival (EHR-FS). Clinical and imaging follow-up data was used as the reference standard.Included in this retrospective study were all consecutive patients with CLM treated with percutaneous or open thermal ablation therapy who had a pre-treatment baseline peak, SULmax, SULmean , and cSULmean are associated with a significant better NHR-FS, independent of the lesion size and number and prior chemotherapy.Fifty-four patients with 90 lesions were selected. Univariable cox regression analysis resulted in eight models. Multivariable analysis revealed that after adjusting for lesion size and the approach of the procedure, none of the metabolic parameters were associated with LTP-FS or EHR-FS. Percutaneous approach was significantly associated with a shorter LTP-FS. It was demonstrated that lower values of SUL18F-FDG PET/CT and the LTP-FS. However, low values of the metabolic parameters were significantly associated with improved NHR-FS. The clinical implication of these findings might be the identification of high-risk patients who might benefit most from adjuvant or combined treatment strategies.We found no association between the metabolic parameters on pre-ablation The online version of this article (doi:10.1007/s00259-017-3637-0) contains supplementary material, which is available to authorized users. In recent years, many new treatment modalities have been established in the field of liver-directed oncologic interventions. Local tumour destruction by means of thermal ablation therapy has emerged as a safe and effective treatment modality for patients with colorectal liver metastases (CLM) who are not surgical candidates . RadiofrAlthough ablation therapy has been proven to be effective in many cases, recurrent disease is a frequently reported problem that jeopardizes patients’ prognosis , 5. Recu18F-fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) for selection and staging of patients who are considered for ablative treatment of liver lesions [18F-FDG PET/CT has only been investigated in terms of detection of hepatic and extrahepatic lesions for staging of patients or for treatment evaluation. However, studies have shown that 18F-FDG PET specific parameters may have a prognostic role in assessing survival of patient with primary or secondary liver malignancies undergoing curative treatment [18F-FDG uptake in tumour lesions [Several studies have investigated the role of  lesions . The rolreatment . This as lesions –12.Up to now, the role of metabolic parameters as imaging biomarkers for recurrent disease following ablative therapy has not been investigated. However, imaging biomarkers can offer a risk stratification tool to identify high-risk patients and allow individualized treatment strategies in order to improve patients’ survival. The aim of this retrospective cohort study was to evaluate the role of metabolic parameters in predicting the prognosis in terms of LTP, intrahepatic, and extrahepatic recurrent disease in patients with CLM treated by means of curative-intent thermal ablation therapy. We hypothesized that an aggressive tumour lesion was associated with a higher metabolic activity, resulting in a higher chance of recurrent disease.18F-FDG PET/CT scan performed within 2 months prior to ablation therapy was an inclusion criterion and all ablation procedures were performed in a curative setting. We excluded tumour lesions that were not assessable on the baseline 18F-FDG PET/CT based on visual inspection. The decision for treatment was made during multidisciplinary tumour-board meeting consisting of at least one experienced liver surgeon, an intervention radiologist, a medical oncologist and a nuclear physician. The main indications for thermal ablation therapy were surgical resection  technically not possible due to multilobar disease or previous liver resection; patient comorbidities could not ensure a safe surgical procedure; and patient preference for minimally invasive treatment. For each procedure, data on clinical and procedural characteristics were collected. Given prior reports of lower 18F-FDG uptake in mucinous type colorectal carcinoma [This retrospective study was reviewed by the institutional review board, and the requirement to obtain informed consent was waived. We retrospectively identified and included all consecutive patients with CLM treated with percutaneous or open RFA or MWA  in Antoni van Leeuwenhoek Hospital Amsterdam, between January 2008 and July 2015. A baseline arcinoma , data onPercutaneous thermal ablation procedure was performed under epidural anesthesia using CT-guidance for needle positioning and evaluation of the ablation zone. The open procedure was performed under general anesthesia, using ultrasound guidance for needle positioning. Until 2010, for RFA, the systems used were Covidien Cool-Tip RF Ablation system with switching controller. Thereafter, a Cool-Tip RF Ablation E-series system  was used. From August 2012, thermal ablation was also performed by means of MWA using an Emprint ablation system . In case of RFA, either a single or cluster antenna was used depending on the required ablation volume. Thermal ablation procedure was performed by two experienced interventional radiologists and according to international guidelines  ensuring18F-FDG PET/CT within 6–8 weeks in case of open ablation procedure. Subsequently, imaging evaluation by means of MRI, CT, or 18F-FDG PET/CT took place every 3 months until 1-year follow-up and thereafter biannually. All patients were followed at least 12 months after the ablation procedure for detection of recurrent disease. After that, patients were followed until the last follow-up visit or death. The follow-up time was defined as the time between the date of the ablation procedure and the development of recurrent disease or the last date on which follow-up was performed. Overall survival (OS) was defined as interval from ablation to death.The standard imaging follow-up schedule consisted of a tri-phasic CT scan within one month in case of percutaneous approach or a 18F-FDG PET was performed in one institute on two Gemini TOF scanner  at 2 min per bed position in 3-dimensional mode. Both scanners were EARL compliant. A graph presenting the recovery curves is included in the supplemental material  based dosage scheme. The dose varied between 190 and 240 MBq depending on a BMI higher or lower than 28. In patients with very high or low BMI, the administered dose was in consultation with the physician. Scanning commenced 60 min after administration (mean 66 +/− 14 min).Whole-body peak, SULmean, partial volume corrected SULmean (cSULmean), SULmax , and the total lesion glycolysis (TLG) were collected for all target lesions. The method for partial volume correction is previously described by Hofheinz et al. [The quantitative analysis was performed in concordance with the PET Response Criteria in Solid Tumours (PERCIST 1.0)  Calculatz et al. .bckgr) was measured by placing a 3-cm diameter spherical volume of interest (VOI) in the healthy liver tissue; the tumour SULpeak was determined in a 1-cm3 spherical VOIpeak. According to the PERCIST 1.0 criteria, a baseline tumour SULpeak has to be ≥1.5 times the SULbckgr of liver tissue plus two times its standard deviation (SD), otherwise quantification can be unreliable. Still, due to the limited number of patients, all measurements were included in the published analysis, also if the tumour SULpeak was below this threshold. A segregated data analysis was performed for measurements that adhered to the strict PERCIST 1.0 criteria . However, this approach sometimes resulted in visually inaccurate delineations, especially when VOI70 was much smaller than the VOI1.5bckgr+2SD. In these cases, metabolic tumour volume was redefined as SULbckgr plus two times SD (n = 4 cases). The mean tumour SUL (SULmean) is a direct derivative of this metabolic tumour volume. For the TLG, VOI threshold was defined as SULbckgr plus two times SD (VOIbckgr+2SD) as recommended by the PERCIST 1.0. The latter threshold was not used for the entire dataset since this threshold frequently resulted in inclusion of a large part of the healthy liver tissue in the VOI, especially when SULpeak was much larger than the VOI1.5bckgr+2SD. Visual inspection was performed by comparing the VOI with the lesion margins as detected on a recently performed contrast enhanced CT. Partial volume correction was implemented for each measurement to account for underestimation of the SUL values. For patient-based analysis, the most metabolically active tumour lesion was used as target lesion.The threshold for the metabolic tumour volume delineation was set at 70% of the tumour SULRecurrent disease was stratified in site of recurrence: LTP, intrahepatic, and extrahepatic recurrence. LTP was the primary endpoint of the study and defined as appearance of tumour foci at the edge of the ablation zone (up to 1 cm from the edge), after at least one contrast-enhanced follow-up study has documented adequate ablation and an absence of viable tissue in the target tumour surrounding ablation margin . Accordi18F-FDG PET/CT) and clinical data were used as the reference standard. The reports of all post-ablation imaging and the scans were evaluated prospectively by the local investigator in order to evaluate the presence of LTP, NHR, or EHR. In case of ambiguity between the imaging reports and the images, the scans were discussed with an experienced intervention radiologist (W.P.) in order to reach consensus. The evaluation of follow-up imaging was blinded for data of the baseline 18F-FDG PET/CT scan.Follow-up imaging , NHR-free survival (NHR-FS), and the EHR-free survival (EHR-FS) were calculated using Kaplan-Meier method. For LTP-FS, analysis was performed based on a per-lesion basis and the NHR-FS and EHR-FS analyses were performed on a per-patient basis where the target lesion was defined as the lesion with the highest SUL18F-FDG uptake of tumour lesions, a subset analysis was performed in the chemo-naïve patients. Chemo-naïve was defined as no chemotherapy at least one year prior to ablation therapy.Univariable cox regression analysis was performed for each metabolic parameter as a risk factor associated with LTP-FS, NHR-FS, or EHR-FS. The metabolic parameter of interest was entered in a multivariable model if the P-value was ≤0.25. Multivariable cox regression analysis was undertaken to evaluate the prognostic potential of the metabolic parameter of interest for prediction of LTP-FS, adjusted for potential effect modifiers such as tumour size, approach of the procedure (open vs. percutaneous), and type of ablation therapy (RFA vs. MWA). Similar analysis was performed for NHR-FS and EHR-FS, adjusted for tumour number and size. The inclusion of covariates in the model was based on clinical relevance. The event per variable rule was used to decide on the appropriate number of variables in the model . In ordeThe proportional hazard assumption for the cox regression model was tested by means of a goodness-of-fit test using chi-square statistics computed for each variable in the model, and adjusted for the other variables in the model. To address the problem of multicollinearity, a correlation coefficient matrix was calculated, with values >0.8 suggesting collinearity between independent variables. In order to obtain clinical relevant results, the median value of the metabolic parameters in the cohort was initially used to retrieve a cut-off value for each parameter. In case of LTP-FS, the median values were based on a per-lesion analysis and in case of NHR-FS and EHR-FS, the median values were based on a per-patient analysis. The multivariable cox regression analysis was performed again including the dichotomized variables. Differences in the survival of the two groups , adjusted for covariates in the model was demonstrated using Kaplan-Meier curves along with the 1-year survival rates. Statistical analyses were performed using RStudio version 3.1.2 open-source software. A P-value <0.05 was considered as statistically significant.peak ≥1.5 times the SULbckgr of liver tissue plus two times its SD). This resulted in 47 patients for the NHR-FS analysis and 38 patients for the EHR-FS analysis according to PERCIST 1.0 . Lesion-based analyses resulted in a LTP rate of 46.5% (40 out of 86 lesions). Of these, 36 out of 86 (41.9%) were identified within 1 year and 20 out of 86 (23.3%) were identified within 6 months following the ablation procedure. The 6-month and 1-year LTP-FS were 80.2% (72.2–89.1) and 58.1% (48.3–69.7), respectively. The median number of follow-up moments within 1-year was 4.5 (range 3–8) in patients that did not develop LTP and 4 (range 2–8) in patients with LTP within 1 year. Thirty-two patients developed NHR (59.0%) and 24 out of 43 patients (56.0%), developed EHR during the course of the disease. The 1-year and 3-year NHR-FS and EHR-FS were 47.4% (35.7–63.0), 37.1% (25.4–54.0) and 62.6% (49.7–79.0), 41.9% (29.1–60.4), respectively. Twenty-one patients (39%) died during the entire follow-up. The median OS was 49.3 months with a 1-year and 5-year survival rate of 94.1% (87.9–100.0) and 28.0% (13.6–57.6), respectively.The univariable analysis resulted in three models for LTP-FS analysis, four models for NHR-FS and one model for EHR-FS Tables  and 3. TP <0.01), independent of the lesion size. Models 4–7 demonstrated that low values of SULpeak, SULmax, SULmean , and cSULmean are significantly associated with a better NHR-FS, independent of the lesion size and number . Based on the hazard ratio and the significance level, the cSULmean did not seem to be superior compared to the SULmean . Kaplan-Meier survival curves were plotted for NHR-FS stratified for high and low SULpeak, SULmax , and SULmean, adjusted for tumour number and size  had received systemic therapy at least 1 year prior to ablation therapy. The univariable and multivariable analysis was repeated in the chemo-naïve patients  was used. The use of different thresholds might have introduced bias in the reported results. However, the segregated analysis for measurements according to the PERCIST 1.0 criteria showed similar results as in the entire dataset. Also, the VOI70 threshold for tumour volume delineation was not according to EANM guidelines [There are limitations to this study. The retrospective nature introduces the risk of selection bias. However, we used clearly defined inclusion and exclusion criteria and included all consecutive patients to mitigate this problem. The threshold for the metabolic tumour volume delineation was set at 70% of the tumour SULidelines . The posidelines 18F-FDG PET/CT. The call for more individualized approach to cancer treatment challenges future studies to investigate prospectively whether risk stratification of patients by means of metabolic parameters will lead to change of management and significant improvement in patients’ survival. Moreover, it should be recognized that the benefit of PET imaging can be extended by using different tracers for different biological features such as hypoxia level, which in turn is associated with negative effect on prognosis [There is cumulative evidence on the prognostic significance of pretreatment rognosis –39. Perh18F-FDG PET/CT in the staging and evaluation setting of patients with CLM. We found no association between the metabolic parameters on pre-ablation 18F-FDG PET/CT and the LTP-FS. However, low values of the metabolic parameters were significantly associated with improved NHR-FS. In the era of tailored approach to patient care, the clinical implication of these findings is the identification of patients who might benefit most from adjuvant or combined treatment strategies.Our findings add to the growing knowledge of the value of ESM 1(DOC 36 kb)ESM 2(DOC 34 kb)ESM 3(DOC 44 kb)Below is the link to the electronic supplementary material.


In line with a hypothesized decrease in inflammatory processes resulting from the yoga and meditation practices, we found that the plasma level of the anti-inflammatory cytokine Interleukin-10 was increased and the pro-inflammatory cytokine Interleukin-12 was reduced after the retreat. Contrary to our initial hypotheses, plasma levels of other pro-inflammatory cytokines, including Interferon Gamma (IFN-γ), Tumor Necrosis Factor (TNF-α), Interleukin-1β (IL-1β), Interleukin-6 (IL-6), and Interleukin-8 (IL-8) were increased after the retreat. Given evidence from previous studies of the positive effects of meditative practices on mental fitness, autonomic homeostasis and inflammatory status, we hypothesize that these findings are related to the meditative practices throughout the retreat; however, some of the observed changes may also be related to other aspects of the retreat such as physical exercise-related components of the yoga practice and diet. We hypothesize that the patterns of change observed here reflect mind-body integration and well-being. The increased BDNF levels observed is a potential mediator between meditative practices and brain health, the increased CAR is likely a reflection of increased dynamic physiological arousal, and the relationship of the dual enhancement of pro- and anti-inflammatory cytokine changes to healthy immunologic functioning is discussed.Thirty-eight individuals (mean age: 34.8 years old) participating in a 3-month yoga and meditation retreat were assessed before and after the intervention for psychometric measures, brain derived neurotrophic factor (BDNF), circadian salivary cortisol levels, and pro- and anti-inflammatory cytokines. Participation in the retreat was found to be associated with decreases in self-reported anxiety and depression as well as increases in mindfulness. As hypothesized, increases in the plasma levels of BDNF and increases in the magnitude of the cortisol awakening response (CAR) were also observed. The normalized change in BDNF levels was inversely correlated with BSI-18 anxiety scores at both the pre-retreat ( Decades of research have linked meditation-based practices to positive psychological and health outcomes in both clinical and nonclinical populations  is a neuroregulatory modulator that has received a paucity of attention in meditation research to date. BDNF is a key neurotrophin that promotes development, survival and plasticity of neurons in the central and peripheral nervous systems , IL-10, and psychological variables which included mindfulness, absorption, depression, anxiety and physical complaints. Finally, we investigated the relationship between psychological improvements and biological changes.Isha retreat center in the southern US. A subset of 98 participants (mean age: 34.8 years old) agreed to participate in the acquisition of blood and salivary markers and are reported upon here. Due to limited funding, only a subset of participants’ samples was analyzed—38 of the participant ID’s were chosen randomly with the only precondition that half be male (n = 19) and half female (n = 19). Of these 38, five participant’s plasma data were not used due to the likelihood of acute illness  based on highly elevated intake (pre-retreat sample) IL-6 levels >8 pg/ml. Seven participant’s saliva data were not used due to extreme outliers of one or more of the six samples collected. An overlapping total of N = 26 subjects had usable pre- and post-blood and salivary samples. This study was carried out in accordance with the recommendations of Quorum Independent Review Board with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Quorum Independent Review Board. Participants filled out a standard consent form detailing the procedure involving providing pre and post saliva and blood samples as well as psychometric questionnaires as well as their option to discontinue at any time.Women and men participants were tested at the beginning and at the end of a 3-month retreat that occurred at the Shoonya meditation (sitting practice focused on a state of “non-doing”), Samyama meditation (sitting practice with breath-focused open awareness) as well as diverse Hatha yoga (mindful movement/stretching) and pranayama (focused breath control) practices. The frequency of such practice prior to beginning the retreat was collected prior to the retreat. As a group, participants reported an average of about 2 h  daily practice, and an average of about 4.5 years  of regular yoga and meditation practice; see Demographics  for about 1–2 h daily. For the last 6 weeks in place of Samyama, Shoonya and kriya practices, participants practiced a form of meditation referred to as Linga sanchalana, which is a focused attention meditation practice, for approximately 1 h twice daily. Finally, in addition to seated meditation practices, participants also practiced Hatha yoga ~1–2 h daily and chanting ~1 h daily. In sum, participants practiced approximately 2 h of sitting meditation practices, 1–2 h of yoga practice with a meditative component and 1 h of chanting daily.The 3-month residential retreat involved daily meditation and yoga practices and was accompanied by a vegetarian diet and a full daily schedule; participants did not engage in their normal work activities, but did work on projects around the retreat center. For the first 6 weeks of the retreat all participants practiced “During the first week of the retreat, participants reported pre-retreat  psychometric measures and gave pre-retreat salivary and blood samples. During the last week they reported post-retreat  psychometric measures and gave post-retreat salivary and blood samples. Specifically, they gave a morning fasting blood sample on retreat day 3, and then again gave a morning fasting blood sample on retreat day 87. On these same days, participants filled out pre-retreat psychometric questionnaires (day 3) and post-retreat psychometric questionnaires (day 87). They also produced six salivary samples at specific time points as described below on retreat day 4, and then again gave six salivary samples at the same time points during the last week of the retreat on retreat day 88.Psychometric questionnaires included the Brief Symptom Inventory-18 , and then throughout the day at the following time points: 30 min post-awakening (5:15 AM), 9:15 AM during the break in between yoga practices and meditation, 12:30 PM just before lunch, 5:10 PM during a break, and lastly at 8:30 PM after meditation, just prior to bed. Saliva of each participant was collected in their mouth and then passively dropped into a Salimetrics saliva collection tube . The tubes from T1 at the beginning of the retreat were then placed in a freezer at −20°C. The same procedure was followed at T2 during the last week of the retreat. Two days after the collection of the T1 and T2 saliva samples, the tubes from all subjects were placed on dry ice and sent to the UC San Diego Clinical Research Biomarker Laboratory. Upon arrival to the lab, all saliva samples, were immediately stored at −80°C and saliva cortisol levels were determined by commercial ELISA . Intra- and inter-assay coefficients were <8%.g for 15 min at 4°C. The supernatant was then aspirated and placed into labeled microcentrifuge tubes (3 cc each) on dry ice immediately. Both T1 and T2 samples were kept on dry ice in labeled trays overnight and sent the next day by overnight delivery to the UC San Diego Clinical Research Biomarker Laboratory for quantitation. Upon arrival to the lab, samples were immediately stored at −80°C. T1 and T2 samples were subjected to analysis simultaneously and in parallel, approximately 2 months after T2 data collection. Plasma levels of BDNF, IFN-γ, TNF-α, IL-1β, IL-6, IL-8, IL-10 and IL-12 were all determined by commercial ELISA kits . Intra- and inter-assay coefficients were <5%.BDNF and inflammatory biomarker levels were assessed in plasma by obtaining EDTA blood from participants early in the morning after awakening and prior to breakfast, on day 4 (T1) of the 90-day program and again on day 88 (T2) of the 90-day program. For both T1 and T2 samples participants were instructed not to engage in any exercise prior to the blood sample acquisition in the morning. The blood was drawn in 10 cc EDTA (purple top) tubes and the tubes were placed on ice for up to 30 min then spun at 400× post hoc analysis to analyze any significant interactions. For the plasma BDNF and inflammatory biomarker analysis, the plasma values were found to vary significantly from normal distribution, thus values were natural log-transformed to produce more normal distributions. Values greater than three standard deviations from the mean were removed, with the # participants removed ranging from one to three subjects for each of the biomarkers analyzed. For each biomarker, a paired t-test for dependent samples was run on the natural log transformed T1 (pre-retreat) and T2 (post-retreat) plasma levels of the biomarkers to assess for significant differences across time from pre- to post-retreat. To assess effects of gender, age, BMI, and change in BMI an ANOVA was also run on the T1 vs. T2 biomarker levels with these demographic categorical/continuous predictors. A limited number of hypothesized correlations between changes in inflammatory biomarkers, BDNF, and cortisol levels and symptoms of psychological distress as measured by the Brief Symptom Inventory were assessed for significance using basic linear correlation procedures. All statistical analyses were run in Statistica 8.0 .For the salivary cortisol, a 2 × 6 Repeated measures ANOVA was conducted with factors being (pre- vs. post-retreat) × (6 time points in the day). Greenhouse-Geisser corrections for multiple comparisons were run and Tukey t-test analysis of psychometric data (see Table t(33) = 4.66, p < 0.0001) as well as each of the subscores which included depressive (t(33) = 2.84, p < 0.01), anxious (t(33) = 4.22, p < 0.0001), and somatic (t(33) = 4.66, p < 0.0001) symptoms. In addition, there was an increase in mindfulness as assessed by the Freiburg Mindfulness Inventory (t(33) = 4.42, p < 0.0001), and no change on absorption as measures by the Tellegen Absorption Scale (p = 0.4).Paired t-tests comparing pre-retreat data to post-retreat data revealed that while the group was on average at a healthy Body Mass Index (BMI) from the outset, small but significant reductions were observed in BMI (t(35) = 4.37, p < 0.0001). In regards to the plasma biomarker data, paired t-tests on the normalized data, after removing extreme outliers, indicated increases in BDNF (t(31) = −4.03, p < 0.001), IFN-γ (t(31) = −2.62, p < 0.05), TNF-α (t(30) = −6.37, p < 0.000001), IL-1β (t(31) = −2.39, p < 0.05), IL-6 (t(30) = −5.13, p < 0.0001), IL-8 (t(30) = −3.30, p < 0.01), and IL-10 (t(29) = −4.65, p < 0.0001). In contrast, there was a decrease in the level of IL-12 (t(31) = 2.37, p < 0.05).Biological markers analyzed included weight as well as inflammatory biomarkers, BDNF and salivary cortisol. As shown in Table t-test of this data indicated no difference between pre and post retreat samples (p = 0.3). A repeated measures 2 × 6 ANOVA conducted on the salivary cortisol measures with factors “pre vs. post retreat” and “time of day” yielded no main effect of pre vs. post retreat (p = 0.50) but a highly significant main effect time of day  = 89.8, p < 0.0000001), indicating the pronounced circadian variation of cortisol levels across the day. There was also a significant interaction between pre vs. post retreat and time of day  = 3.31, p < 0.01). Post hoc analysis of this significant interaction indicated that at T2, cortisol levels 30 min post-awakening (peak cortisol level associated with the CAR) was significantly higher in the post-retreat compared to the pre-retreat samples .A calculation of the area under the curve of the salivary cortisol concentration across the day was done and a paired  day . In addition, these demographic characteristics were entered as predictors into the ANOVA analyses of the effects on the plasma inflammatory biomarkers and BDNF levels. Here we found significant interactions only between gender and three of the dependent variables: BDNF, IFN-γ and IL-10. For BDNF there was a main effect of gender such that women were found to demonstrate higher levels of plasma BDNF than men  = 6.88, p < 0.05) and there was a nearly significant gender × pre-post interaction (p = 0.06) indicating a tendency for women to show greater relative increases in BDNF over the course of the retreat. For IFN-γ a significant gender × pre-post interaction was obtained  = 4.33, p < 0.05) and post hoc analysis indicated that the increases in IFN-γ levels were significant for women (p < 0.01) but not men (p = 1). For IL-10 a significant gender × pre-post interaction was obtained  = 4.90, p < 0.05) and post hoc analysis indicated that the increases in IL-10 levels were significant for women (p < 0.001) but not men (p = 0.31). There was no significant interaction between participant age, gender, or BMI and change in the other biomarker levels.Including BMI, change in BMI, age and gender as covariates in the ANOVA of the salivary cortisol data , revealed no significant interaction between these demographic characteristics and the significant effect on the salivary CAR. There was, however, a significant main effect of gender on salivary cortisol levels such that women showed higher overall cortisol levels across the day than men  and post-retreat  such that those with greater anxiety scores tended to evidence a smaller pre- to post-retreat increase in plasma BDNF levels. No such correlations were observed between other BSI-18 measures or mindfulness scores and BDNF levels and no correlations were obtained between these psychometric measures and CAR findings. A relationship was found between the magnitude of the pre-retreat CAR and the resultant change in BDNF levels, however this relationship was found to be driven by one particular outlier so this relationship was taken to be non-significant. Lastly, in order to assess the unexpected effect demonstrating increases in both the prototypical anti-inflammatory marker IL-10 and the range of pro-inflammatory markers, correlations were run between IL-10 levels and the other interleukins. We found a positive correlation between the change in IL-10 levels and the change in IL-12 levels from pre- to post-retreat, signifying that the participants who demonstrated greater increases in anti-inflammatory IL-10 levels tended to also demonstrate greater increases in pro-inflammatory IL-12 levels.To explore the possible relationship between the changes in biomarker levels among each other and related to psychological changes, a small number of Pearson product-moment correlations between inflammatory markers, CAR dynamics, BDNF plasma levels and psychological measures were executed. The normalized change in BDNF plasma level  was inversely correlated with BSI-18 anxiety scores at both the pre-retreat  tended to demonstrate a less robust increase in BDNF levels, consistent with the notion that BDNF levels were indeed related to psychological wellness. This fits with evidence from others suggesting that BDNF is negatively correlated with depression  levels is diet, which has a profound impact on the gut microbiota community and resultant microbiome signaling processes within the brain and body. Diet was controlled during the retreat in that participants were fed high-quality vegetarian food prepared by the staff. Studies in mice have demonstrated associations between the gut microbiota and BDNF in the regulation of brain physiology, behavior, and memory . In addition, further controls would allow for understanding of the role of other contextual factors  affecting the expression and regulation of these biological processes. Future research that assesses broad panels of biomarkers and global studies with more appropriate control populations are needed to clarify the extent to which these changes are related specifically to the meditation and yoga practices, and given the suggestive findings reported here such further work is warranted.BRC: designed study, analyzed results and wrote the manuscript. MSG: analyzed results and wrote the manuscript. RM: organized data collection and analyzed results. CTP: wrote the manuscript. PJM: designed study, organized data quantitation and edited the manuscript.This study received funding from co-author RM. RM has no financial relationship with Isha Yoga and had the following involvement with the study: Organized data collection, analyzed results. The corresponding BC and senior PM authors had full executive control over and ownership of the data, processing of biological samples, final analysis, and interpretation of results, as well as the write-up and decision to publish. PM is the Scientific Director of the Chopra Foundation. The Chopra Foundation is a 501 (c) (3) organization dedicated to improving health and well-being partly through research on yoga and meditation and is connected with the Chopra Center that offers meditation and yoga courses.


A growing number of investigations of AFP as a tumour-specific biomarker have concluded that AFP is an important target for cancer treatment. AFP also plays an immunomodulatory role in the treatment of several autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, myasthenia gravis and thyroiditis. In an effort to support biochemical screening and drug design and discovery, we attempted to express and purify human AFP in a Bac-to-Bac system. Two key factors affecting the expression of recombinant human AFP (R-AFP), namely the infectious baculovirus inoculum volume and the culturing time post-infection, were optimized to maximize the yield. We achieved a high yield of approximately 1.5 mg/l of harvested medium with a 72–96 h incubation period after infection and an inoculum volume ratio of 1:100. We also assessed the role of R-AFP in the proliferation of the human liver cancer cell line Bel 7402, and the results indicated that R-AFP promoted the growth of hepatoma cells. We concluded that this method can produce high yields of R-AFP, which can be used for studies related to AFP. The albuminoid genes evolved from a common ancestor and exhibit considerable similarity in their primary structure. For example, human AFP and human serum albumin (HSA) share 40% identity with highly conserved cysteine residues. Human AFP consists of 609 amino acids, has a molecular mass of 69 kDa and contains only one glycosylation site (N233). However, the glycosylation site may link various carbohydrate moieties, and the structure of the carbohydrate moieties varies in different tissues and diseases  and 150 mM NaCl) and 80 μl of nickel (Ni)-charged resin . After the sample was mixed and shaken for 2 h, the 6× His-tag R-AFP in the supernatant was captured by Ni-charged resin and was eluted with 100 μl of 300 mM imidazole in HBS buffer.The eluted R-AFP was analysed by SDS/PAGE. The reduced protein SDS/PAGE sample contained a reducing buffer, such as DTT, and was boiled for 3 min. The non-reduced protein sample did not contain a reducing buffer and was not heated .Cytoplasmic R-AFP was analysed as follows: after 72 h incubation, the media were harvested and centrifuged at 3000 rpm for 15 min. Cell pellets were resuspended in 40 ml of HBS buffer, sonicated on ice for 15 min with 3 s/9 s intervals and then centrifuged at 13000 rpm for 60 min. The supernatant was collected, and supernatant containing R-AFP protein was further purified and analysed to determine the expression level of secreted R-AFP as described recently .After 72 h incubation, the media (approximately 1 litre) containing the secreted R-AFP were harvested and centrifuged at 4000 rpm for 10 min, the supernatant was collected and filtered with a 0.45 µm filter membrane . The supernatant was then concentrated to 100 ml by cross-flow filtration , and the buffer was changed to HBS buffer. The concentrate was centrifuged at 10000 rpm for 30 min, and the supernatant was collected and passed through Ni-charged resin . R-AFP was captured by the resin and eluted with 300 mM imidazole in HBS buffer. The eluted buffer was concentrated to 1 ml using a 10 kDa filter tube  and then further purified by gel filtration chromatography using a Superdex 200 column . The detection wavelength was 280 nm. Elution was performed with an HBS buffer at a flow rate of 1 ml/min .Human cord blood AFP was precipitated by ammonium sulfate and passed through an anti-AFP affinity chromatography column. AFP-positive fractions were collected and concentrated. The purity of prepared AFP was 92.7% as determined by SDS/PAGE. The protein was stored at −80°C until use .Separation of protein: The target protein was electrophoretically separated by SDS/PAGE. Electrotransfer: Proteins were transferred from the gel to a PVDF membrane in a constant voltage of 60 V for 2 h at 4°C. Immunodetection: Blocking buffer (3%) was added to the PVDF membrane, and the membrane was rocked gently for 2 h. Then, the membrane was rinsed with TBST buffer three times. The appropriate concentration of anti-AFP antibody was added, and the membrane was rocked gently for 12 h at 4°C. The appropriate concentration of horseradish peroxidase-conjugated anti-rabbit antibody was added, and the membrane was rocked gently for 1 h at 37°C. The membrane was then washed with TBST buffer, developing reagent was added, and development was monitored. The antibody against AFP and the horseradish peroxidase-conjugated anti-rabbit antibody were purchased from Sangon Biotech Co., Ltd. (Shanghai) and Jackson ImmunoRes Lab, Inc., (U.S.A.) respectively [ectively ,7,8.The optimization experiments were designed by comparing the effects of two major factors (the infectious baculovirus inoculum volume and the post-infection time) on the production of R-AFP. These experiments were performed by infecting 1-litre cell cultures with baculovirus and incubating the cells at a constant temperature of 27°C under shaking conditions (110 rpm) –18. We hThe monoclonal antibody against human AFP (anti-AFP) was prepared according to standard procedures . BrieflyExpression of the human AFP receptor (AFPR) in the Bel 7402 cell line was examined using a laser confocal microscope as described previously . Briefly2 for 48 h. The supernatant was removed and replaced with 150 μl of fresh medium without FBS for another 24 h. Different concentrations of extracted human AFP (E-AFP), R-AFP (0–80 mg/l), HSA or anti-AFP were added to each well for 24 h and then pulsed with 1 mCi of 3H-TdR. The cells were harvested on to a glass microfibre filter 4 h later using a multiple sample harvester. The incorporation of 3H-TdR was measured using an LKB 1209 Rackbeta liquid scintillation counter. We used E-AFP as a positive control. To determine whether the influence of human AFP on proliferation was specific, the blockage of anti-AFP and HSA as structural analogues was also assessed, and a control group without AFP or HSA was also paralleled to be done, according to the previously described procedure [Bel 7402 cells were suspended in RPMI-1640 medium containing 10% FBS and were added to a 24-well plate at 1 ml per well followed by incubation at 37°C in a humidified atmosphere of 5% COrocedure .4 Bel 7402 cells per well were plated in 96-well plates and cultured in RPMI-1640 medium supplemented with 10% FBS at 37°C in a humidified atmosphere of 5% CO2 for 48 h. The cultured cell medium was replaced with medium without FBS for another 24 h, and the cells were treated with human AFP (20 mg/l) for 48 h. The effects of E-AFP and R-AFP on cell growth were measured by MTT assay as described in a previous study [A490 − treated group A490)/control group A490 × 100%.A total of 1.5 × 10us study . The groafp vector could be digested with BamHI and HindIII restriction enzymes to release a band measuring approximately 2000 bp, confirming the presence of the recombinant human afp gene. DNA sequencing of recombinant pFastBac 1–afp confirmed the existence of the coding sequence in the correct frame of the vector, without any mutation or alteration in the afp gene sequences. afp vector and confirmed that the correct homologous recombination occurred in the bacmid.The expression vectors were confirmed by agarose gel electrophoresis. afp gene. The molecular mass of the cytoplasmic R-AFP bands (lanes 3 and 4) was similar to that of the secreted R-AFP bands, but the bands were faint and impure. These results indicated that the cytoplasmic R-AFP concentration was lower than the extracellular R-AFP concentration in the medium and that cytoplasmic R-AFP may bind with other proteins. The R-AFP protein was further evaluated by Western blotting (Secreted R-AFP (in the insect medium) and cytoplasmic R-AFP (in the lysed insect cells) were assessed by SDS/PAGE, and the results are shown in blotting B. The mePurification of the secreted R-AFP in the medium is straightforward. First, the medium was concentrated by buffer exchange using HBS buffer. In the second step, R-AFP in the medium was captured on Ni-charged resin, eluted with 300 mM imidazole and purified by gel filtration chromatography. The eluted chromatography position of R-AFP is shown in 7 pfu/ml) and the same Sf9 cell density (2 × 106 cells/ml). The infectious baculovirus inoculum volumes  and R-AFP production were monitored at different post-infection times . R-AFP protein expression reached the highest level (approximately 1.5 mg/l of medium) at a 1:100 volume of the infectious baculovirus inocula when harvested at 96 h post-infection, the expression of R-AFP in 1:100 volume of the infectious baculovirus inocula has statistical difference compared with 1:500 volume groups (P<0.05) (In the process of analysing R-AFP expression, it was found that different conditions led to different expression levels. 3H-TdR incorporation results indicated that after treatment with R-AFP or E-AFP, the content of 3H-TdR in Bel 7402 cells was markedly enhanced (3H-TdR content in the Bel 7402 cells was markedly enhanced in a dose-dependent manner. The peak of the increase reached 123.0% compared with the control group (To detect the bioactivity of R-AFP, in the present study, we selected the human liver cell line Bel 7402 to examine the effects of R-AFP on cellular proliferation. First, we observed the expression of AFPR, and the results revealed that Bel 7402 expressed AFPR and that the receptor was located in the cell membrane A. Secondol group D. In addol group E. These E. coli expression systems, in yeast and in the milk of transgenic goats, but these expression systems produced low amounts of human AFP. For example, human AFP expressed in E. coli was deposited in bacterial inclusion bodies and subjected to harsh denaturants [R-AFP expressed in the Bac-to-Bac system is soluble and stable as a monomer, and its bioactivity is similar to human AFP extracted from blood. Its IP is pH 4.5. To extract human AFP, a great deal of human cord blood is needed, which is difficult to purify. Expressing R-AFP in the Bac-to-Bac system allows a large amount of purified bioactive protein to be obtained without the need for human cord blood. R-AFP may allow the study of the biochemical features of human AFP and drug screening. Human AFP expression was previously reported in aturants ,11,21. Haturants . The puraturants . There iaturants . The R-ATo optimize the yield of the R-AFP protein, the post-infection time was considered. Recombinant protein was expressed intracellularly and needed time to be modified and transported to the extracellular space. Thus, the best harvest time depends on the protein and the experiment . In the 2+ to stimulate the transduction of cellular signals to stimulate N-ras and c-myc expression, leading to promote proliferation of NIH 3T3 cells and Bel 7402 cells [The effects of extracellular human AFP on cellular proliferation are mainly achieved through signal transduction mediated by AFPR. Previously, we found that Bel 7402 cells express AFPR . In the 02 cells ,22. In t02 cells  and phos02 cells . These i02 cells ,25. In c


In cancer immunotherapy, biomarkers are important for identification of responsive patients. This study was aimed to find biomarkers that predict clinical outcome of WT1 peptide vaccination.Candidate genes that were expressed differentially between long- and short-term survivors were identified by cDNA microarray analysis of peripheral blood mononuclear cells that were extracted from 30 glioblastoma patients (discovery set) prior to vaccination and validated by quantitative RT-PCR using discovery set and different 23 patients .SDC-4 mRNA expression levels distinguished between the long- and short-term survivors: 1-year survival rates were 64.0 and 18.5% in SDC4-low and -high patients, respectively.SDC-4 is a novel predictive biomarker for the efficacy of WT1 peptide vaccine. SDC-4 as a biomarker to predict clinical outcome using peripheral blood mononuclear cells obtained from patients with glioblastoma, a malignant brain tumor, who were treated with WT1 peptide vaccine. With 30 samples of peripheral blood mononuclear cells prior to vaccination, 32 candidate genes were filtrated by microarray, and finally only SDC-4 was validated by RT-PCR using another 23 samples. Accordingly, 1-year survival rates were 64.0 and 18.5% in SDC4-low and -high patients, respectively.Recently, cancer immunotherapies are becoming a standard therapeutic option. To improve their efficacy, identification of biomarkers is important to select responsive patients. In this study, we identified  WT1 gene is overexpressed in leukemia and a variety of solid tumors, in which it exerts an oncogenic function [WT1 mRNA-electroporated [WT1, a transcription factor, regulates many kinds of important genes that play important roles in embryogenesis, cell proliferation and differentiation . The WT1function ,5. WT1, function . Our grofunction  or WT1 moporated  dendritioporated , acute moporated , chronicoporated ,21, myeloporated , multiploporated , malignaoporated , infantioporated , and lunoporated , breast oporated , pancreaoporated ,30, ovaroporated ,32, uteroporated ,33 and soporated ,35.GBM is a malignant brain tumor with very poor prognosis. The standard therapy for the newly diagnosed GBM is surgery, followed by irradiation and chemotherapy. However, a 5-year survival rate is less than 10%, and once recurrence occurs, therapeutic options are limited . In 2008In order to improve the clinical usefulness of WT1 peptide vaccine, it is crucial not only to biologically enhance the vaccine's efficacy but also to select patients who are likely to respond to the vaccine. Identification of responders would be facilitated by the availability of reliable biomarkers that predicted the clinical outcome of patients treated with the WT1 peptide vaccine.To date, several studies by our group and others have identified markers that are correlated with the clinical efficacy of WT1-based immunotherapy. Malignant glioma patients with higher WT1 expression levels (score 3–4) lived longer after immunotherapy than those with lower expression levels (score 1–2) . An incrSDC-4 mRNA expression levels prior to WT1 vaccination were a promising predictive biomarker for clinical outcome in these patients.In this study, we used cDNA microarrays to comprehensively analyze gene-expression profiles of peripheral blood mononuclear cells (PBMCs) with the goal of identifying biomarkers that predicted the outcome of WT1 peptide vaccination in patients with recurrent or conventional therapy-resistant GBM. The results revealed that HLA-A*24:02, of the natural WT1–235 peptide . The binding affinity of mWT1–235 for HLA-A*24:02 is higher than that of nWT1–235, and mWT1–235 induced a much stronger CTL response against WT1-expressing tumor cells. GMP-grade WT1 peptide was purchased from Multiple Peptide Systems  and Peptide Institute  as the lyophilized peptide.The WT1 peptide vaccine consists of WT1-CTL epitope peptide and Montanide ISA51 adjuvant. The peptide used in this study is a modified 9-mer WT1 peptide , in which M was replaced by Y at the second amino acid position, an anchor position for Sixty patients were enrolled in a Phase II study of WT1 peptide vaccination of GBM patients, the result of which we reported previously ,13. PatiPBMC samples obtained prior to WT1 vaccination were available from 53 of the 60 patients enrolled in the Phase II study; all 53 were used in this study. Thirty of the patients were randomly assigned to the discovery set, and the remaining 23 were assigned to the validation set.The median OS of the patients in the discovery set was 347 days from the start of the vaccination and 460 days from the time of recurrence or disease progression to death or censored time points. The median PFS, which was defined as the days from the start of the vaccination to the disease progression, was 62 days. In the microarray analysis, to select differentially expressed genes (DEGs) that influence not only short-term outcome  but also long-term outcome, the 30 patients in a discovery set were divided into two groups with OS from the time of recurrence or disease progression. The 15 patients with OS of ≥460 days were defined as long-term survivors , whereas the remaining 15 patients with OS of <460 days were defined as short-term survivors  .In the verification process using discovery set, OS was the days from the first vaccination to death or censored time points which should more strictly reflect the vaccination effect. Thus, in the verification step, patients who survived ≥347 days (the median OS of the discovery set according to the immediately preceding definition) were defined as long-term survivors, whereas those who survived <340 days were defined as short-term survivors.In the validation set, median OS from the first vaccination was 257 days. The 12 patients who survived ≥256 days were defined as long-term survivors, and the 11 patients who survived <256 days were defined as short-term survivors.Supplementary Table 1). The Phase II study of WT1 peptide vaccination and cDNA microarray analysis of the blood samples was approved by the ethical review board of Osaka University Hospital.All short-term survivors in the discovery and validation sets died before a censored time point. Therefore, patient categorization of long- and short-term survivors was fixed  criteria. When clinical response was obvious, WT1 vaccination was continued at 2-week intervals for the next few months, and then at 1–3-month intervals until obvious tumor progression or deterioration of the patient's condition was observed.Peripheral blood was obtained from patients immediately before the first vaccination, followed by separation of PBMCs by density gradient centrifugation using Lymphocyte Separation Solution . Separated PBMCs were stored in liquid nitrogen prior to use.Frozen PBMCs were thawed, and total RNA was isolated using the TRIzol reagent , purified using chloroform and subjected to isopropanol and ethanol precipitation. Purified total RNA was quantitated using a NanoDrop ND-1000 .RNAs from PBMCs of the discovery set patients were sent to Toray Industries , which performed RNA-based cDNA microarray analysis using Human oligo chip 25k ver1.00 (Toray Industries).10 of Welch's t-test between the two groups (y-axis) versus log2(fold change) (x-axis) was made [2. DEGs were selected using this volcano plot according to the following conditions: y > |x|−1, |x| >0.5 and y >1.0. To enrich for DEGs, correlations between individual gene expression intensities and either PFS or clinical responses according to RECIST criteria were examined by Pearson's and Spearman's correlation coefficient, respectively. Genes that did not exhibit significant p-values  were excluded from further analysis. In this series of microarray analyses, a nonstringent cut-off for the p-value was set according to MAQC guidelines [10 was greater than |log2(fold difference)|–1; third, |log2(fold difference)| was greater than 0.5; fourth, -log10  greater than 1.0; fifth, p-value of Pearson's correlation between PFS and signal intensities less than 0.1; and sixth, p-value of Spearman's correlation between clinical outcome and signal intensities less than 0.2.Microarray analysis data were transformed using global normalization, followed by quantile normalization . First, was made . The folidelines . FinallySupplementary Table 2. Target sequences are available from the NCBI nucleotide database [ACTB, GAPDH, RPL13, RPL18A, PPIA, TSR2 and RNA28S1. Each control gene was validated in comparison with each other, and correlation matrix was generated, followed by scoring each gene. Consequently, ACTB was determined as the most stable internal control gene among all the candidate control genes and was used as the internal control. Then, ΔCTs were calculated as . All data concerning gene expression levels were converted to log2(1 + 2−ΔCT), and the relative expression levels were statistically analyzed.RNAs from PBMCs were reverse-transcribed into first-strand cDNA using SuperScript VILO cDNA Synthesis Kit and Master Mix (Life Technologies). Sequences of primers used for RT-PCR are provided in database . Sample Cox proportional hazard regression models were used to evaluate associations between patient characteristics and OS in the discovery and validation sets. In the microarray analysis, PFS was logarithmically transformed to yield a normal distribution.In statistical analysis of the result from RT-PCR, the Jarque–Bera test was used to assess normality of variable distributions, and the F-test was used to test homogeneity of variance.To promote statistical power, parametric analyses were conducted as much as possible. For examination of the difference between long- and short-term survivors, an appropriate transformation  was performed for individual genes to satisfy the requirement for normality. All normally distributed variables were checked for homogeneity of variance and analyzed by Student's t-test. Non-normally distributed variables were analyzed using the Mann–Whitney U-test.SDC-4 expression levels that discriminated responders and nonresponders  were determined using a receiver operating characteristic (ROC) curve in order to maximize the Youden's index (defined as Youden's J statistic = sensitivity + specificity -1). The ROC curve was drawn based on the cut-off value of OS, which maximized area under the ROC curve (AUC). The horizontal and vertical axes indicate 1-specificity and sensitivity for responder, respectively. Sensitivity, specificity, positive and negative predictive values, and accuracy of the prediction of OS by the SDC-4 expression levels were calculated using standard formulas. Kaplan–Meier curves and a two-sided log-rank test were used to assess differences between two groups defined by the SDC-4 cut-off value. Statistical analysis was conducted using appropriate software including JMP Pro 10.0  and R-commander [The ommander .Patients with recurrent or conventional therapy-resistant GBM who were treated with WT1 peptide vaccine were selected as the cohort for this analysis. PBMCs were obtained prior to WT1 vaccination from 53 of 60 patients in this vaccination trial. The strategy to search useful biomarkers for prediction of clinical outcome is shown schematically in 10 p-value) versus log2 (fold change), in which each dot indicates one of a total of 25,000 genes, is shown in –1, |x| > 0.5 and y > 1.0. To further enrich for candidate genes, the correlation between the signal intensities of individual genes and either PFS or clinical response was evaluated by Pearson's correlation and Spearman's rank correlation analysis, respectively, and genes with p-values ≥ 0.1 (Pearson's) or ≥ 0.2 (Spearman's) were excluded. Ultimately, 32 genes were selected as candidates  correlated with OS in the validation set  and nonresponders (OS of <256 days) was determined to be 0.001 according to the AUC-maximized ROC curve using the Youden's index; SDC-4 expression levels ≤0.001 and >0.001 predicted responders and nonresponders, respectively. Prediction of likely responders had 70.4% sensitivity, 76.0% specificity, 76.0% positive prediction value, 70.4% negative prediction value and 73.1% accuracy; in addition, a Chi-square test demonstrated a significant difference (p < 0.001).First, based on the values of OS and ion sets A. GeneraSDC-4 expression level (0.001). The patients were divided into two groups, SDC-4-low (SDC-4 ≤ 0.001) and -high (SDC-4 > 0.001) groups  groups B. The diectively B.SDC-4 expression levels and baseline patient characteristics, including the two factors mentioned above, were assessed using Cox proportional hazard regression models in all the 53 patients (Supplementary Table 3). This analysis allowed us to assess the association between SDC-4 expression levels and the risk of death at any given time points while controlling for other predictors that may affect the risk of death. As a result, it was suggested that high SDC-4 expression level of SDC-4 was a significant risk factor for the worse clinical outcome  and a predictor of the clinical outcome independent of other risk factors.As shown in SDC-4 expression levels in PBMCs obtained prior to WT1 vaccination were significantly and negatively correlated with OS of recurrent or conventional therapy-resistant GBM patients who were treated with WT1 peptide vaccine, and that SDC-4 expression levels were a useful biomarker for prediction of clinical outcome.In this study, we demonstrated that T cells are a major player in tumor immunity. T cells are activated through an interaction between T-cell receptors and antigen/MHC molecule complexes on antigen-presenting cells (APCs); that is regulated in a costimulatory or coinhibitory manner by accessory receptors. A costimulatory signal is transmitted by the interaction between CD28 receptor on T cells and CD80 or CD86 on APCs. On the other hand, coinhibitory signals are transmitted by a variety of molecules, including the interactions among CTLA-4 (cytotoxic T lymphocyte antigen-4) on T cells for CD80 and CD86 on APCs; programmed cell death-1 (PD-1) and its ligands PD-L1 and PD-L2; B- and T-lymphocyte attenuator and herpes virus entry mediator; Tim-3 (T cell immunoglobulin- and mucin domain-containing molecules 3) and Tim-3L ,44; and in vitro and exacerbated contact hypersensitivity responses in vivo [-/- T cells into sublethally γ-irradiated allogeneic mice induces hyperproliferation of infused T cells [+ monocytes inversely correlate with allostimulatory capacity, and knockdown of DC-HIL enhances allostimulation. Deletion of DC-HIL abrogates the T-cell suppressor activity of myeloid-derived suppressor cells (MDSCs) [Syndecans are type-I transmembrane heparan sulfate proteoglycans that bind to the extracellular matrix and a variety of cytokines, chemokines and growth factors, thereby modifiying their local concentration, stability and accessibility to their respective receptors; consequently, they significantly influence cell proliferation and differentiation. Mammals have four known syndecans, , of which only SDC-4 is expressed ubiquitously; expression of the other three is tissue-restricted . SDC-4 i in vivo . Consist T cells . Collect T cells . DC-HIL  (MDSCs) . These o (MDSCs) .SDC-4 in PBMCs, which correlated with favorable clinical effects of WT1 peptide vaccination of GBM patients, reflected downregulated expression of SDC-4 on T cells. In addition, this downregulation of SDC-4 augmented WT1-specific immune responses to WT1 peptide vaccination by attenuating the interaction between SDC-4 and DC-HIL on monocytes (DCs), leading to improved clinical effects.Based on the findings described above, our present results might be interpreted as follows. Low expression of Helicobacter pylori infection [On the other hand, it was also reported that, besides T-cell activation state, various pathophysiological conditions, including bacterial endotoxin shock , acute pnfection , atherosnfection  and ischnfection , are assSupplementary Table 3, low-performance status is a common risk factor for poor prognosis of GBM, and the past chemotherapy might dampen immunological competence that supports WT1 peptide-based immunotherapy. On the other hand, although old age is a common risk factor for poor prognosis of GBM as well as low-performance status, no association between OS and age was detected. Since WT1 immunotherapy is a very mild therapy without significant adverse effects (only skin erythema at the vaccine injection sites) that give the organ damages, almost all patients, regardless of ages, are tolerable to the WT1 immunotherapy and may be effective to immunotherapy. On the other hand, since chemotherapy has strong adverse effects, the patients become nontolerable to it as the patients’ ages increase, resulting in the decrease in clinical outcome. Therefore, age is not important prognostic factor in immunotherapy, but PS, which reflects the patients’ immune conditions, is a very important prognostic factor. Repeated chemotherapy gives the damages to the immune system and thus decreases the clinical effects of the following immunotherapy. Therefore, the results that previous chemotherapy but not age is a bad prognostic factor are reasonable.Regarding the two risk factors in LRRN3, PCDH17, HIST1H4C and PGLYRP1, but not SDC-4, as biomarkers that discriminated between long- (>900 days) and short-term (≤900 days) survivors. The discrepancy in the markers identified in our study and theirs, which used a different peptide vaccine, implies that different targeted TAAs lead to different immune responses in patients. This might mean that these different immune responses are dependent upon whether T cells, DCs and MDSCs or other cell types are major players in the immune reaction, and that the major players are determined by differences in the targeted TAAs and/or types of malignancies.To our knowledge, only one previous study  involvedSDC-4 as a biomarker were divided into the two: discovery and validation processes. The candidate genes identified in the discovery process using 30 patients were validated in the next process using different 23 patients in a validation set, resulting in the identification of SDC-4 alone as a biomarker to predict clinical outcome of WT1 peptide vaccine. We wish to perform a prospective study composed of a larger sample size in the near future, and then we may obtain a more definite finding that supports the conclusion of the present study. In addition, a large sample size-based study may give us an opportunity to find another promising biomarker for WT1 peptide vaccination, and a combination of SDC-4 and the other biomarkers may become a more precise predictor for clinical outcome of the therapy.Since sample size of the present study was not so large, the processes to identify the SDC-4 expression level in PBMCs prior to vaccination was a prognostic factor for long-term survivors. This result was compatible with previously reported immune-suppressive functions of SDC-4 expressed on T cells. These findings suggested that SDC-4 might be useful not only as a biomarker to select patients who would respond to the immunotherapy but also as a target to improve the effect of the immunotherapy.In this study, among patients with glioblastoma who received WT1 peptide vaccine, low WT1 overexpresses in leukemia and a variety of solid tumors and performs an oncogenic function, and WT1 protein is one of the most superior pan-tumor-associated antigens.Glioblastoma multiforme (GBM) is a malignant brain tumor with very poor prognosis, and once recurrence occurs, therapeutic options are limited.In 2008, we reported the promising results of a Phase II clinical study of WT1 vaccination for recurrent or conventional therapy-resistant GBM patients.In order to improve the clinical usefulness of WT1 peptide vaccine, it is important not only to enhance clinical efficacy but also to select the responders to WT1 peptide vaccine.Novel methods to predict responders prior to WT1 vaccination were awaited.Peripheral blood mononuclear cells from 30 GBM patients prior to WT1 vaccination in a discovery set were subjected to cDNA microarray analysis, candidate genes that differentially expressed between long- and short-term survivors were selected, followed by verification with quantitative RT-PCR.The filtrated candidate genes were validated for the correlation between gene expression levels and clinical effects using different 23 GBM patients of a validation set.Thirty-two differentially expressed genes (DEGs) were extracted by cDNA microarray analysis using 30 GBM patients in a discovery set, and 15 DEGs were verified by quantitative RT-PCR.SDC-4 was identified as a biomarker for prediction of overall survival (OS).The 15 DEGs were validated using 23 patients in a validation set, and only SDC-4 that discriminated between OS of ≥256 days (responders) and OS of <56 days (nonresponders) was 0.001, and the SDC-4 expression levels of ≤0.001 and >0.001 predicted responders and nonresponders, respectively, with 70.5% sensitivity and 76.0% specificity.Cut-off value of mRNA expression levels of SDC-4-low and -high patients, respectively.One-year OS rates were 64.0 and 18.5% in SDC-4 is a biomarker to predict clinical outcome for GBM patients treated by WT1 peptide vaccine.Click here for additional data file.Click here for additional data file.Click here for additional data file.


Current evidence supports the applicability of the Leeds Sleep Evaluation Questionnaire (LSEQ) in screening for insomnia. The psychometric properties of the LSEQ have never been investigated in an African population. Therefore, this study aimed to validate the adapted version of the LSEQ-Mizan (LSEQ-M) in Ethiopian university students.2) from Mizan-Tepi University, Mizan-Aman, South-west Ethiopia completed the LSEQ-M, the General Anxiety Disorder Scale-7 and a semi-structured questionnaire for socio-demographics. Insomnia was screened in accordance with the International Classification of Sleep Disorders as a measure of concurrent validity.Of a preliminary sample of 750 (random sampling), 424 students (age = 21.87 ± 4.13 years and body mass index = 20.84 ± 3.18 kg/mp < 0.0001). The original 4-Factor model was valid in Ethiopian university students for screening for insomnia.Although, individual items showed ceiling and floor effect, the LSEQ-M as a scale did not have these effects. Good internal consistency  and strong internal homogeneity as measured by the correlation coefficient between items scores and the LSEQ-M global score was found. The LSEQ-M showed excellent screening applicability for insomnia with optimal cut-off scores of 52.6 , and the area under the curve, 0.95  contains supplementary material, which is available to authorized users. The growing endemicity of sleep disorders is becoming a health concern around the globe –4. The sSleep disturbances in Ethiopian university students are related to insomnia and its associated conditions i.e. problems in sleep onset, short sleep duration and poor sleep quality , 3. EthiThe Leeds Sleep Evaluation Questionnaire (LSEQ) is a widely used tool for the diagnosis of sleep disorders including insomnia , 14. The2). Self-reported problems with memory was the exclusion criteria. The purpose and procedures of the study were explained to the volunteers in detail. The university students comprised of many ethnicities, some of them had limited reading proficiency level of the national language i.e. Amharic [A sample of 750 students was selected by simple random sampling method across Mizan campus of the Mizan-Tepi University, Mizan-Aman town, Bench Maji Zone, Southwest, Ethiopia. Four hundred and twenty four completed the cross-sectional study i.e. provided filled in answers for LSEQ, Generalized Anxiety Disorder Scale-7 (GAD-7), sub-structured questionnaire for socio-demographics, and participated in a clinical interview. The majority of the participants were males (82.5%), and young adults , quality of sleep (QOS), ease of awakening from sleep (AFS) and alertness and behavior following wakefulness (BFW) . The iteAn experienced sleep researcher blinded to the LSEQ-M score clinically interviewed all the participants who completed the study. The presence of insomnia was determined according to the International Classification of Sleep Disorders, revised criteria (ICSD-3) , 19. TheThe statistical analysis was performed using SPSS version 16.0  along with AMOS . Internal consistency was assessed by the Cronbach alpha test, while internal homogeneity was tested by Pearson’s correlation analysis between LSEQ-M items and the LSEQ-M global scores. Discriminative validity was assessed by independent t-test for LSEQ-M item as well as the LSEQ-M global score. Diagnostic validity was evaluated by receiver operating curve (ROC) analysis. ICSD-3 based screening for primary insomnia by sleep expert served as the gold standard and the LSEQ-M global score was the test variable . Area un2 = 29.59). Twenty two outliers were deleted for factor analysis with Χ2 > 29.59 [Multivariate outliers were estimated by calculation of Mahalanobis distance (criterion of a = .001 with 10 df (number of variables), the critical Χ > 29.59 . There w > 29.59 . None of > 29.59 . ExploraConfirmatory factor analysis (CFA) was performed using maximum-likelihood extraction. The factor loadings (standardized estimates) of the LSEQ-M items on the latent factors were calculated. The CFA was run on six models of the LSEQ-M Table ; 1-Factop < 0.01) (Table The socio-demographics of the Ethiopian university students participating in the study are given in Table 1) Table .Table 1SThe groups of Ethiopian university students identified as normal and with moderate anxiety levels based on GAD-7 evaluation differed across the LSEQ-M total score, as well as scores of all the items score except the item-9 Table . The diaThe sample satisfied the conditions for factor analysis as indicated by the results of Kaiser-Meyer-Olkin test of sampling adequacy, Bartlett’s test of sphericity, anti-image matrix Table  and commp-value; none of the models had absolute fit to the data  among those with moderate level of anxiety as measured by the GAD establish the diagnostic known-group or discriminative validity of the tool in this population of Ethiopian university students Table . InsomniThe diagnostic validity of the scale against ICSD-3 criteria for insomnia in this sample of Ethiopian university students was in an excellent range . Few stuThe results of the EFA were inconclusive, but the outcome of CFA favored the original 4-Factor model of the LSEQ-Min the Ethiopian university students Tables , 6 and 7Biased gender ratio in the study sample and non-application of objective measurement of sleep i.e. polysomnography, actigraphy are important limitations. The gender ratio in the study sample was 0.22, although it is 0.55 in the university students. Female students were less likely to complete the clinical interview, which might have resulted in gender bias. The test re-test reliability and inter/intra-rater reliability were not assessed. Future studies should look into this aspect. The merits include validation of a tool in a population, which has high prevalence of sleep problems but does not have access to advanced sleep medicine professionals and/or facilities.The study findings suggest that LSEQ has favorable psychometric validity than the PSQI in Ethiopians. The LSEQ-M was found to be a valid tool for screening for insomnia in this sample of Ethiopian university students.Additional file 1: (SAV 30 kb)